Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.

Incretin hormones exert pleiotropic metabolic actions beyond the pancreas. Although the heart expresses both incretin receptors, the cardiac biology of GIP receptor (GIPR) action remains incompletely understood. Here we show that GIPR agonism did not impair the response to cardiac ischemia. In contrast, genetic elimination of the Gipr reduced myocardial infarction (MI)-induced ventricular injury and enhanced survival associated with reduced hormone sensitive lipase (HSL) phosphorylation; it also increased myocardial triacylglycerol (TAG) stores. Conversely, direct GIPR agonism in the isolated heart reduced myocardial TAG stores and increased fatty acid oxidation. The cardioprotective phenotype in Gipr-/- mice was partially reversed by pharmacological activation or genetic overexpression of HSL. Selective Gipr inactivation in cardiomyocytes phenocopied Gipr-/- mice, resulting in improved survival and reduced adverse remodeling following experimental MI. Hence, the cardiomyocyte GIPR regulates fatty acid metabolism and the adaptive response to ischemic cardiac injury. These findings have translational relevance for developing GIPR-based therapeutics.

Therapy with anti-PD-L1 immune check-point inhibitors is approved for several cancers, including advanced urothelial carcinomas. PD-L1 prevalence estimates vary widely in bladder cancer, and lack of correlation between expression and clinical outcomes and immunotherapyresponse may be attributed to methodological differences of the immunohistochemical reagents and procedures. We characterized PD-L1 expression in 235 urothelial carcinomas including 79 matched pairs of primary and metastatic cancers using a panel of four PD-L1 immunoassays in comparison with RNAscope assay using PD-L1-specific probe (CD274). The antibody panel included three FDA-approved clones (22C3 for pembrolizumab, 28.8 for nivolumab, SP142 for atezolizumab), and a commonly used clone E1L3N. Manual scoring of tissue microarrays was performed in each of 235 tumors (624 tissue cores) and compared to an automated image analysis. Expression of PD-L1 in tumor cells by ≥1 marker was detected in 41/142 (28.9%) primary tumors, 13/77 (16.9%) lymph nodes, and 2/16 (12.5%) distant metastases. In positive cases, high PD-L1 expression (>50% cells) was detected in 34.1% primary and 46.7% metastases. Concordant PD-L1 expression status was present in 71/79 (89.9%) cases of matched primary and metastatic urothelial carcinomas. PD-L1 sensitivity ranked from highest to lowest as follows: RNAscope, clone 28.8, 22C3, E1L3N, and SP142. Pairwise concordance correlation coefficients between the four antibodies in 624 tissue cores ranged from 0.76 to 0.9 for tumor cells and from 0.30 to 0.85 for immune cells. RNA and protein expression levels showed moderate to high agreement (0.72-0.87). Intra-tumor expression heterogeneity was low for both protein and RNA assays (interclass correlation coefficients: 0.86-0.94). Manual scores were highly concordant with automated Aperio scores (0.94-0.97). A significant subset of 56/235 (23.8%) urothelial carcinomas stained positive for PD-L1 with high concordance between all four antibodies and RNA ISH assay. Despite some heterogeneity in staining, the overall results are highly concordant suggesting diagnostic equivalence of tested assays.

The brain transforms the need for water into the desire to drink, but how this transformation is performed remains unknown. Here we describe the motivational mechanism by which the forebrain thirst circuit drives drinking. We show that thirst-promoting subfornical organ neurons are negatively reinforcing and that this negative-valence signal is transmitted along projections to the organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO). We then identify molecularly defined cell types within the OVLT and MnPO that are activated by fluid imbalance and show that stimulation of these neurons is sufficient to drive drinking, cardiovascular responses, and negativereinforcement. Finally, we demonstrate that the thirst signal exits these regions through at least three parallel pathways and show that these projections dissociate the cardiovascular and behavioral responses to fluid imbalance. These findings reveal a distributed thirst circuit that motivates drinking by the common mechanism of drive reduction.

Early life stress (ELS) in the form of child abuse/neglect is associated with an increased risk of developing social dysfunction in adulthood. Little is known, however, about the neural substrates or the neuromodulatory signaling that govern ELS-induced social dysfunction. Here, we show that ELS-induced downregulation of dopamine receptor 3 (Drd3) signaling and its corresponding effects on neural activity in the lateral septum (LS) are both necessary and sufficient to cause social abnormalities in adulthood. Using in vivo Ca2+ imaging, we found that Drd3-expressing-LS (Drd3LS) neurons in animals exposed to ELS show blunted activity in response to social stimuli. In addition, optogenetic activation of Drd3LS neurons rescues ELS-induced social impairments. Furthermore, pharmacological treatment with a Drd3 agonist, which increases Drd3LS neuronal activity, normalizes the social dysfunctions of ELS mice. Thus, we identify Drd3 in the LS as a critical mediator and potential therapeutic target for the social abnormalities caused by ELS.

Adult neural stem cells (NSCs) derive from embryonic precursors, but little is known about how or when this occurs. We have addressed this issue using single-cell RNA sequencing at multiple developmental time points to analyze the embryonic murine cortex, one source of adult forebrain NSCs. We computationally identify all major cortical cell types, including the embryonic radial precursors (RPs) that generate adult NSCs. We define the initial emergence of RPs from neuroepithelial stem cells at E11.5. We show that, by E13.5, RPs express a transcriptional identity that is maintained and reinforced throughout their transition to a non-proliferative state between E15.5 and E17.5. These slowly proliferating late embryonic RPs share a core transcriptional phenotype with quiescent adult forebrain NSCs. Together, these findings support a model wherein cortical RPs maintain a core transcriptional identity from embryogenesis through to adulthood and wherein the transition to a quiescent adult NSC occurs during late neurogenesis.

Long non-coding RNAs (lncRNAs) may contribute to carcinogenesis and tumor progression by regulating transcription and gene expression. The role of lncRNAs in the regulation of thyroid cancer progression is being extensively examined. Here, we analyzed three lncRNAs that were overexpressed in papillary thyroid carcinomas, long intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR, ROR) PVT1 oncogene (PVT1), and HOX transcript antisense intergenic RNA (HOTAIR) to determine their roles in thyroid tumor development and progression. ROR expression has not been previously examined in thyroid carcinomas. Tissue microarrays (TMAs) of formalin-fixed paraffin-embedded tissue sections from 129 thyroid cases of benign and malignant tissues were analyzed by in situ hybridization (ISH), automated image analysis, and real-time PCR. All three lncRNAs were most highly expressed in the nuclei of PTCs. SiRNA experiments with a PTC cell line, TPC1, showed inhibition of proliferation with siRNAs for all three lncRNAs while invasion was inhibited with siRNAs for ROR and HOTAIR. SiRNA experiments with ROR also led to increased expression of miR-145, supporting the role of ROR as an endogenous miR-145 sponge. After treatment with TGF-β, there was increased expression of ROR, PVT1, and HOTAIR in the PTC1 cell line compared to control groups, indicating an induction of their expression during epithelial to mesenchymal transition (EMT). These results indicate that ROR, PVT1, and HOTAIR have important regulatory roles during the development of PTCs.

Abstract

AIM:

To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB.

METHODS:

Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects on food intake and glucose regulation. The effect of pro-UGN or UGN overexpression, using adeno-associated virus (AAV) vectors, was assessed in diet-induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas.

RESULTS:

GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV-mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice.

CONCLUSION:

GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes.

Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1-/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres.

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.

Oxytocin (OXT) receptors (OXTRs) are prominently expressed in hippocampal CA2 and CA3 pyramidal neurons, but little is known about its physiological function. As the functional necessity of hippocampal CA2 for social memory processing, we tested whether CA2 OXTRs may contribute to long-term social recognition memory (SRM) formation. Here, we found that conditional deletion of Oxtr from forebrain (Oxtr-/-) or CA2/CA3a-restricted excitatory neurons in adult male mice impaired the persistence of long-term SRM but had no effect on sociability and preference for social novelty. Conditional deletion of CA2/CA3a Oxtr showed no changes in anxiety-like behavior assessed using the open field, elevated plus maze and novelty-suppressed feeding tests. Application of a highly selective OXTR agonist [Thr4,Gly7]-OXT to hippocampal slices resulted in an acute and lasting potentiation of excitatory synaptic responses in CA2 pyramidal neurons that relied on N-methyl-D-aspartate receptor activation and calcium/calmodulin-dependent protein kinase II activity. In addition, Oxtr-/- mice displayed a defect in the induction of long-term potentiation, but not long-term depression, at the synapses between the entorhinal cortex and CA2 pyramidal neurons. Furthermore, Oxtr deletion led to a reduced complexity of basal dendritic arbors of CA2 pyramidal neurons, but caused no alteration in the density of apical dendritic spines. Considering that the methodologies we have used to delete Oxtr do not rule out targeting the neighboring CA3a region, these findings suggest that OXTR signaling in the CA2/CA3a is crucial for the persistence of long-term SRM.

SIGNIFICANCE STATEMENT: Oxytocin receptors (OXTRs) are abundantly expressed in hippocampal CA2 and CA3 regions, but there are little known about their physiological function. Taking advantage of the conditional Oxtr knockout mice, the present study highlights the importance of OXTR signaling in the induction of long-term potentiation at the synapses between the entorhinal cortex and CA2 pyramidal neurons and the persistence of long-term social recognition memory. Thus, OXTRs in the CA2/CA3a may provide a new target for therapeutic approaches to the treatment of social cognition deficits, which are often observed in patients with neuropsychiatric disorders.

The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NF-κB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NF-κB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell-intrinsic responses that include proliferation, migration and invasion. In addition, CBM-dependent activation of NF-κB elicited cancer cell-extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NF-κB signaling in AGTR1+ breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II-dependent NF-κB signaling pathways to improve the treatment of this breast cancer subset.